Hydrolysis and assay of triglycerides

Abstract

A method and composition for the hydrolysis and assay of triglycerides are disclosed. The method includes the steps of adding lipase and cholesterol esterase to a triglyceride in combination with a glycerol assay system and determining the amount of triglycerides present based on the amount of glycerol produced. The composition includes a mixture of lipase, cholesterol esterase and a glycerol assay system.

What is claimed is: 
     
       1. A method for determining the amount of triglycerides present in an aqueous fluid which comprises contacting the fluid with a mixture of a lipase and cholesterol esterase for a time sufficient to hydrolyze the triglyceride to glycerol and free fatty acids and determining the amount of triglycerides present based on the amount of glycerol produced. 
     
     
       2. A method as claimed in claim 1 wherein the lipase is produced by a microorganism selected from the group consisting of Rhizopus delemar, and Chromobacterium viscosum. 
     
     
       3. A method as claimed in claim 1 wherein the cholesterol esterase is obtained from the microorganism Pseudomonas aeruginosa or from beef pancreas. 
     
     
       4. A method as claimed in claim 1 wherein the lipase is produced from a microorganism selected from the group consisting of Rhizopus delemar, Rhizopus arrhizus and Chromobacterium viscosum and the cholesterol esterase is obtained from the microorganism Pseudomonas aeruginosa or from beef pancreas. 
     
     
       5. A method as claimed in claim 1 wherein the amount of glycerol present is determined enzymatically. 
     
     
       6. A method as claimed in claim 1 wherein the hydrolysis is carried out at a pH of from 6 to 9 and a temperature of from 20° to 40° C. 
     
     
       7. A method as claimed in claim 1 wherein a buffer is added to the mixture of lipase and cholesterol esterase. 
     
     
       8. A method as claimed in claim 1 wherein a surfactant is added to the mixture of lipase and cholesterol esterase. 
     
     
       9. A method as claimed in claim 1 wherein the fluid is a body fluid. 
     
     
       10. A method as claimed in claim 9 wherein the body fluid is serum or plasma. 
     
     
       11. A method as claimed in claim 1 wherein there is present from 0.01 to 5.0 U cholesterol esterase per 10 U lipase. 
     
     
       12. A method as claimed in claim 1 where there is present from 0.35 to 1.8 U cholesterol esterase per 10 U lipase. 
     
     
       13. A method as claimed in claim 1 wherein the amount of glycerol present is determined by contacting the liberated glycerol with a glycerol assay system comprising: adenosine triphosphate; phosphoenolpyruvate; pyruvate kinase; lactate dehydrogenase; reduced nicotinamide adenine dinucleotide; glycerol kinase and a magnesium salt. 
     
     
       14. A method as claimed in claim 1 wherein the amount of glycerol present is determined by contacting the liberated glycerol with a glycerol assay system comprising: adenosine triphosphate; glycerol kinase; α-glycerolphosphate dehydrogenase; oxidized nicotinamide adenine dinucleotide; diaphorase and a dye. 
     
     
       15. A composition for the determination of triglycerides by enzymatic hydrolysis comprising a mixture of lipase, cholesterol esterase and a glycerol assay system. 
     
     
       16. A composition as claimed in claim 15 wherein there is present a buffer. 
     
     
       17. A composition as claimed in claim 15 wherein there is present a surfactant. 
     
     
       18. A composition as claimed in claim 15 wherein the assay system comprises: adenosine triphosphate; phosphoenolpyruvate; pyruvate kinase; lactate dehydrogenase; reduced nicotinamide adenine dinucleotide; glycerol kinase and a magnesium salt. 
     
     
       19. A composition as claimed in claim 15 wherein the assay system comprises: adenosine triphosphate; glycerol kinase; α-glycerol phosphate dehydrogenase; oxidized nicotinamide adenine dinucleotide; diaphorase and a dye. 
     
     
       20. A test device for the determination of triglycerides in a fluid which comprises a carrier incorporated with lipase, cholesterol esterase and a glycerol assay system.